Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

Abstract

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.

Keywords: Arabidopsis thaliana; cuticle integrity; organ separation; pectin methylesterification; stem growth.

Figure 1. Growth phenotypes and leaf cuticle characteristics of Arabidopsis thaliana plants overexpressing the PMEI5 gene. WT plants (A) exhibited straight stems, while PMEI OE plants (B) had twisted stems and stunted growth. The WT plants (C, F) showed clear organ separation; in PMEI OE plants (D, G, and H) the side stems carrying a leaf or inflorescence failed to separate from the main stem. This led to a thickened stem and twisting growth around the areas of failed separation. Note that the stem regions associated with an unseparated leaf or inflorescence were always on the inside of a twist or loop. (E) Comparison of toluidine blue staining of leaves of WT- (left) and PMEI5 OE- plants (right) to evaluate the integrity of the cuticle. Toluidine blue cannot penetrate an intact cuticle; this dye was readily washed off from the surfaces of the WT leaves (E, left). In PMEI OE leaves (E, right), the staining remained after washing, indicating a cuticle defect. Also note that the cuticle of the WT leaves appeared as a whitish layer under the dissection microscope light.

Figure 2. Stress-strain-curves for stem segments of 3 WT lines as compared with the stem segments of 3 PMEI5 OE lines.

Figure 3. Sugar composition of cell walls of WT- and PMEI5 OE- stems and immunostaining of stem sections to detect homogalacturonans of differing methylesterification. (A) Glycosyl residues in the hot water soluble fraction and (B) residual fraction. The data are based on the average of 5 replicates (+/− SD). An asterisk over a column indicates a significant (p < 0.05, paired t-test) difference between PMEI5 OE and WT. (C) Estimate of esterification levels through quantification of methanol released on saponification. The average of 4 biological replicates (+/− SE) are shown. (D) Representative immuno-staining of sections of stem branching points of the PMEI5 overexpressor (OE) and wild-type (WT) plants. Sections stained with toluidine blue were immunostained with the antibodies 2F4 (binds to homogalacturonan stretches that are largely demethylesterified and cross linked by calcium bridges), JIM5 (binds to highly methylesterified pectin with a loose sequence of methyl groups), and JIM7 (binds to highly methylesterified homogalacturonans with dense stretches of methylesters).